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Xylan utilization in human gut commensal bacteria is orchestrated by unique modular organization of polysaccharide-degrading enzymes.

ABSTRACT: Enzymes that degrade dietary and host-derived glycans represent the most abundant functional activities encoded by genes unique to the human gut microbiome. However, the biochemical activities of a vast majority of the glycan-degrading enzymes are poorly understood. Here, we use transcriptome sequencing to understand the diversity of genes expressed by the human gut bacteria Bacteroides intestinalis and Bacteroides ovatus grown in monoculture with the abundant dietary polysaccharide xylan. The most highly induced carbohydrate active genes encode a unique glycoside hydrolase (GH) family 10 endoxylanase (BiXyn10A or BACINT_04215 and BACOVA_04390) that is highly conserved in the Bacteroidetes xylan utilization system. The BiXyn10A modular architecture consists of a GH10 catalytic module disrupted by a 250 amino acid sequence of unknown function. Biochemical analysis of BiXyn10A demonstrated that such insertion sequences encode a new family of carbohydrate-binding modules (CBMs) that binds to xylose-configured oligosaccharide/polysaccharide ligands, the substrate of the BiXyn10A enzymatic activity. The crystal structures of CBM1 from BiXyn10A (1.8 Å), a cocomplex of BiXyn10A CBM1 with xylohexaose (1.14 Å), and the CBM from its homolog in the Prevotella bryantii B14 Xyn10C (1.68 Å) reveal an unanticipated mode for ligand binding. A minimal enzyme mix, composed of the gene products of four of the most highly up-regulated genes during growth on wheat arabinoxylan, depolymerizes the polysaccharide into its component sugars. The combined biochemical and biophysical studies presented here provide a framework for understanding fiber metabolism by an important group within the commensal bacterial population known to influence human health.

SUBMITTER: Zhang M

PROVIDER: S-EPMC4156774 | biostudies-literature | 2014 Sep

REPOSITORIES: biostudies-literature

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Xylan utilization in human gut commensal bacteria is orchestrated by unique modular organization of polysaccharide-degrading enzymes.

Zhang Meiling M Chekan Jonathan R JR Dodd Dylan D Hong Pei-Ying PY Radlinski Lauren L Revindran Vanessa V Nair Satish K SK Mackie Roderick I RI Cann Isaac I

Proceedings of the National Academy of Sciences of the United States of America 20140818 35

Enzymes that degrade dietary and host-derived glycans represent the most abundant functional activities encoded by genes unique to the human gut microbiome. However, the biochemical activities of a vast majority of the glycan-degrading enzymes are poorly understood. Here, we use transcriptome sequencing to understand the diversity of genes expressed by the human gut bacteria Bacteroides intestinalis and Bacteroides ovatus grown in monoculture with the abundant dietary polysaccharide xylan. The m ...[more]

PMID: 25136124

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Project description:Background and aimsNectar is a very complex mixture of substances. Some components (sugars and amino acids) are considered primary alimentary rewards for animals and have been investigated and characterized in numerous species for many years. In contrast, nectar proteins have been the subject of few studies and little is known of their function. Only very recently have detailed studies and characterization of nectar proteins been undertaken, and then for only a very few species. This current work represents a first step in the identification of a protein profile for the floral nectar of Cucurbita pepo. In this regard, the species studied is of particular interest in that it is monoecious with unisexual flowers and, consequently, it is possible that nectar proteins derived from male and female flowers may differ.MethodsManually excised spots from two-dimensional (2-D) electrophoresis were subjected to in-gel protein digestion. The resulting peptides were sequenced using nanoscale LC-ESI/MS-MS (liquid chromatography-electrospray ionization/tandem mass spectrometry). An MS/MS ions search was carried out in Swiss-Prot and NCBInr databases using MASCOT software.Key resultsTwo-dimensional electrophoresis revealed a total of 24 spots and a different protein profile for male and female flower nectar. Four main proteins recognized by 2-D electrophoresis most closely resemble β-d-xylosidases from Arabidopsis thaliana and have some homology to a β-d-xylosidase from Medicago varia. They were present in similar quantities in male and female flowers and had the same molecular weight, but with slightly different isoelectric points.ConclusionsA putative function for xylosidases in floral nectar of C. pepo is proposed, namely that they may be involved in degrading the oligosaccharides released by the nectary cell walls in response to hydrolytic enzymes produced by invading micro-organisms. Several types of oligosaccharides have been reported to increase the pathogenic potential of micro-organisms. Thus, it is possible that such a mechanism may reduce the virulence of pathogens present in nectar.

Project description:Butyrate produced by gut microbiota has multiple beneficial effects on host health, and oligosaccharides derived from host diets and glycans originating from host mucus are major sources of its production. A significant reduction of butyrate-producing bacteria has been reported in patients with inflammatory bowel diseases and colorectal cancers. Although gut butyrate levels are important for host health, oligosaccharide metabolic properties in butyrate producers are poorly characterized. We studied the metabolic properties of fructooligosaccharides (FOSs) and other prebiotic oligosaccharides (i.e. raffinose and xylooligosaccharides; XOSs) in gut butyrate producers. 1-Kestose (kestose) and nystose, FOSs with degrees of polymerization of 3 and 4, respectively, were also included. Fourteen species of butyrate producers were divided into four groups based on their oligosaccharide metabolic properties, which are group A (two species) metabolizing all oligosaccharides tested, group F (four species) metabolizing FOSs but not raffinose and XOSs, group XR (four species) metabolizing XOSs and/or raffinose but not FOSs, and group N (four species) metabolizing none of the oligosaccharides tested. Species assigned to groups A and XR are rich glycoside hydrolase (GH) holders, whereas those in groups F and N are the opposite. In total, 17 enzymes assigned to GH32 were observed in nine of the 14 butyrate producers tested, and species that metabolized FOSs had at least one active GH32 enzyme. The GH32 enzymes were divided into four clusters by phylogenetic analysis. Heterologous gene expression analysis revealed that the GH32 enzymes in each cluster had similar FOS degradation properties within clusters, which may be linked to the conservation/substitution of amino acids to bind with substrates in GH32 enzymes. This study provides important knowledge to understand the impact of FOS supplementation on the activation of gut butyrate producers. Abbreviations: SCFA, short chain fatty acid; FOS, fructooligosaccharide; XOS, xylooligosaccharide; CAZy, Carbohydrate Active Enzymes; CBM, carbohydrate-binding module; PUL, polysaccharide utilization locus; S6PH sucrose-6-phosphate hydrolase.

Dataset Information

Xylan utilization in human gut commensal bacteria is orchestrated by unique modular organization of polysaccharide-degrading enzymes.

Publications

Xylan utilization in human gut commensal bacteria is orchestrated by unique modular organization of polysaccharide-degrading enzymes.

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